Project Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a major health problem. The Center for Disease Control estimates that approximately 1/3 to 1/4 of the world?s population is latently infected. RNA polymerase (RNAP), the enzyme responsible for all transcription in bacteria, is the target for the Rifamycin (Rif) class of antibiotics, a first line therapeutic treatment for TB. RNAP is thus a proven and attractive target for the development of new drugs. This highlights the importance of our recent structural and functional characterization of Mtb RNAP and the roles of two essential transcription factors required for full transcriptional activity. The previous grant enabled us to provide a 2.8 resolution crystal structure of an RNAP transcription initiation complex (TIC) from M. smegmatis and more recently cryo-EM structures of Mtb transcription complexes. In this proposal, cryo-EM will be used to examine RNAP complexes as a starting point to elucidate the mechanisms of a family of relatively uncharacterized transcription factors, the WhiB factors. The WhiB factors are only found in Actinobacteria and have roles in Mtb that include essentiality for growth and division, and responses to host induced stresses including antibiotic tolerance, nitric oxide, macrophage invasion and reactive oxygen species. We will use a multidisciplinary approach that includes structural, biochemical, genomic and in vivo experiments (in collaboration with J. Rock) to understand the roles and mechanism of this important, but relatively uncharacterized family of transcription factors. The results from the aims here have the potential to not only elucidate the mechanism and biology of these factors, but also provide a platform for new targets for clade-specific antibiotic development and serve to guide us on how to increase the efficacy of the current repertoire of antibiotics. The results from the previous funding period have led to high resolution structures of Mycobacteria RNAP (by cryo-EM and crystallography), and provided the opportunity to characterize how Rif and Rif derivatives that inhibit Rif resistant (RifR) bacteria inhibit Mycobacteria RNAP. Here we propose to continue this line of research with structurally uncharacterized Rif derivatives, provided by S. Brady, that inhibit additional RifR Mtb RNAPs. Clostrioides difficile (Cdiff), a Gram-positive, sporulating, anaerobic bacterium, is an opportunistic pathogen which is deadly to compromised hosts. Fidaxomicin (Fdx), the only other FDA approved antibiotic which targets RNAP, is a powerful treatment for Cdiff infection. Our recent work established that Fdx can inhibit Mtb RNAP potently, but that potency is dependent on the Actinobacteria-specific transcription factor RpbA, which is absent in Cdiff. Here we propose to extend our expertise in biochemical and structural studies of bacterial RNAPs to include the previously uncharacterized clade of Firmicutes to which Cdiff belongs. The results here will elucidate the structural and biochemical basis for Fdx potency as well as provide a structural and biochemical basis for exploiting Cdiff RNAP for drug development and optimization.